Beta Residues Research Articles

Identification and characterization of Mg2+ binding sites in isolated alpha and beta subunits of H(+)-ATPase from Bacillus PS3.

The properties of the nucleotide binding sites in the isolated beta and alpha subunits of H(+)-ATPase from Bacillus PS3 (TF1) have been examined by studying the EPR properties of bound VO(2+), which is a paramagnetic probe for the native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with different mixtures of both alpha and beta subunits, and with the catalytic alpha 3 beta 3 gamma subcomplex have been characterized by a combination of EPR, ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by (51)V hyperfine coupling parameters (A( parallel) = 168 x 10(-)(4) cm(-)(1) and A( perpendicular) = 60 x 10(-)(4) cm(-)(1)) that suggest that VO2+ binds to the isolated beta subunit with at least one nitrogen ligand. Results obtained for the analogous VO2+ complex with the isolated alpha subunit are virtually identical. ESEEM and HYSCORE spectra are also reported and are similar for both complexes, indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A( parallel)((14)N) = 4.44 MHz, and A( perpendicular)((14)N) = 4.3 MHz and quadrupole coupling parameters e(2)()qQ approximately 3.18 MHz and eta approximately 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alpha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ bound to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha 3 beta 3 hexamer has a second nitrogen ligand with magnetic properties similar to those of a histidine imidazole. This situation is analogous to that in the alpha 3 beta 3 gamma subcomplex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S., Sigalat, C., Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 14281-14293]. These data are interpreted in terms of only partially structured nucleotide binding sites in the isolated beta and alpha subunits as compared to fully structured nucleotide binding sites in the alpha 3 beta 3 heterohexamer, the alpha 3 beta 3 gamma subcomplex, and the whole TF1 ATPase.

The Nature of the Catalytic Domain of 2′-5′-Oligoadenylate Synthetases

2'-5'-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induced enzymes that are activated by double-stranded RNA. To understand why, unlike other DNA and RNA polymerases, they catalyze 2'-5' instead of 3'-5' phosphodiester bond formation, we used molecular modeling to compare the structure of the catalytic domain of DNA polymerase beta (pol beta) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary sequence identity is low, like pol beta, P69 can assume an alphabetabetaalphabetabetabeta structure in this region. Moreover, mutation of the three Asp residues of P69, which correspond to the three catalytic site Asp residues of pol beta, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes. Biochemical and mutational studies demonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of the heterodimer was half of that of the wild type homodimer, although the two proteins bound double-stranded RNA and ATP equally well.

Modification of beta 2glycoprotein I by glutardialdehyde. Conformational changes and aggregation accompany exposure of the cryptic autoepitope.

Autoantibodies from patients with antiphospholipid syndrome (APS) recognize an epitope on beta 2glycoprotein I (beta 2 GPI) only when native beta 2 GPI is adsorbed on surfaces composed of anionic phospholipids or oxidized polystyrene. beta 2 GPI was modified with the crosslinking agent, glutardialdehyde (GDA), which induced exposure of the anti-beta 2 GPI epitope at GDA:beta 2 GPI mol ratios in the range of 500-2000. A second crosslinking agent, dimethyl-suberimidate (DMS), did not expose the epitope, which may be a consequence of its having less tendency than GDA to form intermolecular links. SDS-PAGE experiments demonstrate that GDA does promote extensive intermolecular crosslinking of beta 2 GPI, and DMS does not. Formaldehyde also reacts with the lysine residues of beta 2 GPI, but does not expose the epitope. The circular dichroism spectra of native and modified beta 2 GPI confirm that GDA induces changes in conformation that are qualitatively different from those caused by formaldehyde. These data provide evidence that binding of lysine residues is not a sufficient condition for exposure of the autoepitope, and also support the likelihood that anti-beta 2 GPI antibodies bind only to aggregates of the protein. Thus, by synthesizing an active holoantigen of beta 2 GPI, conditions were defined that are necessary for binding of human autoantibodies.

Identification of novel transmembrane gene sequence and its use for cell-surface targeting of beta subunit of human chorionic gonadotropin.

We identified a 685-nucleotide gene fragment that codes for the transmembrane and cytoplasmic domains of glycoprotein of the LEP strain rabies virus and carried out experiments designed to express a novel fusion protein on the cell surface. The cDNA encoding the membrane anchor sequence was fused in the correct reading frame to the 3' end of the cDNA encoding the beta subunit of human chorionic gonadotropin (beta(h)CG), a secretory glycoprotein that is used as an antigen for a contraceptive vaccine being developed in our laboratory. The fusion gene cassette was placed under the control of a vaccinia virus early promoter and cloned in a host-restricted fowlpox viral vector. The recombinants, when used to infect mammalian cells that do not allow the replication of fowlpox virus, expressed the N-terminal 135 amino acid residues of beta(h)CG anchored in the cell membrane by the 75-amino acid C-terminal sequence derived from rabies virus glycoprotein. This hybrid protein is correctly processed post-translationally and transported efficiently to the plasma membrane of non-permissive cells such that the anchored beta(h)CG molecule retains the correctly folded native antigenic epitope(s).

Engineering of betabellin-15d: A 64 residue beta sheet protein that forms long narrow multimeric fibrils

Engineering of betabellin-15d: A 64 residue beta sheet protein that forms long narrow multimeric fibrils

Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.

Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends.

The beta-thymosins are a family of highly polar peptides which serve in vivo to maintain a reservoir of unpolymerized actin monomers. In vitro, beta-thymosins form 1:1 complexes with actin monomers and inhibit both polymerization and exchange of the bound nucleotide. Circular dichroism data indicate that free thymosin beta 4 is predominantly unstructured, containing at most six residues of alpha-helix, and that up to six additional residues may adopt an alpha-helical conformation upon binding actin. NMR data indicate that many parts of thymosin beta 4 are not in tight contact with actin. Contacts between specific residues in actin and thymosin beta 4 were identified by zero-length cross-linking followed by isolation and sequencing of cross-linked peptides. After carbodiimide-mediated cross-linking, Lys-3 of thymosin beta 4 was cross-linked to Glu-167 of actin, and Lys-18 of thymosin beta 4 was cross-linked to one of the the N-terminal acidic residues of actin (Asp-1-Glu-4); the cross-linked actin residues lie within subdomains 3 and 1, respectively. These two contacts flank the alpha-helical region of thymosin beta 4 and place it on the barbed end; thymosin beta 4 can thus block actin polymerization sterically. After transglutaminase-mediated cross-linking, Lys-38 of thymosin beta 4 was cross-linked to Gln-41 of actin, placing the C-terminal region of thymosin beta 4 in contact with subdomain 2 on the pointed end; thymosin beta 4 may sterically block actin polymerization at the pointed end as well as the barbed end of the monomer. The distance between the pointed-end and barbed-end contacts requires that the C-terminal half of thymosin beta 4 be in a predominantly extended conformation.

Functions of conserved tryptophan residues of the core light-harvesting complex of Rhodobacter sphaeroides.

We have examined mutants in the core light-harvesting complex of Rhodobacter sphaeroides in which the tryptophan residues located at positions alpha+11, beta+6, and beta+9 have been mutated to each of the three other aromatic amino acids, namely tyrosine, phenylalanine, and histidine. We confirm that the alpha+11 residue and show that the beta+9 residue each form a hydrogen bond to a C2-acetyl group of a BChl molecule. Mutation of either of these residues to a phenylalanine results in a breakage of the normal hydrogen bond, whereas a histidine in either of these positions is able to form a hydrogen bond to the BChl. Comparison of the absorption spectra with the hydrogen bonding of the C2-acetyl groups for the various mutants demonstrates a role for this molecular interaction in the tuning of the absorption properties of the complex. We further demonstrate that there is a consistent linear relationship between the downshift in the C2-acetyl stretching mode and the red shift in the absorption maximum, in both core and peripheral antenna complexes. This linear relationship allows us to estimate the contribution of H bonding to the red shifts of these complexes. Though the residue beta+6 is found not to be directly involved in interactions with the pigment molecules, mutation of this residue is shown in some cases to result in both a destabilization of the complex and a decrease in the binding site homogeneity. Finally, a consideration of the amount of antenna complex present in the various mutants shows an important role for the reaction center and/or the pufX gene product in the assembly or stabilization of this membrane protein.

Structural requirements of synthetic oligosaccharides to bind monoclonal antibodies against Chlamydia lipopolysaccharide.

Monoclonal antibodies were generated against a synthetic glycoconjugate containing the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo (Kdo, 3-deoxy-D-manno-2-octulopyranosonic acid) which represents the genus-specific epitope of the lipopolysaccharide from the obligatory intracellular human pathogen Chlamydia. Antibodies of all immunoglobulin G isotypes were obtained and characterized by enzyme immunobinding and inhibition assays using the immunizing antigen as well as chemically synthesized derivatives of the Kdo trisaccharide. The latter contained (1) one of the three residues in beta- instead of alpha-linkage, (2) a Kdo residue the carboxyl group of which had been reduced to a CH2OH group (Kdo(C1-red)), or (3) changing the linkage of the terminal Kdo from 2-->8 to 2-->4. Only one compound, namely, alpha-Kdo-(2-->8)-alpha-Kdo(C1-red)-(2-->4)-alpha-Kdo exhibited binding to and inhibition of Kdo trisaccharide-specific antibodies, whereas all other compounds were not active. Structural and conformational investigations using NMR spectroscopy at high field on the allyl glycosides of the oligosaccharides 6-12 confirmed the conformational similarities between those structures 4, 5, and 10 which were able to bind to the antibodies investigated.

Protein-nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double-stranded DNA template-primer.

The conformation of the DNA and the interactions of the nucleic acid with the protein in a complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and 19-mer/18-mer double-stranded DNA template-primer (dsDNA) are described. The structure of this HIV-1 RT complex with dsDNA serves as a useful paradigm for studying aspects of nucleotide polymerases such as catalysis, fidelity, drug inhibition, and drug resistance. The bound dsDNA has a bend of approximately 41 degrees at the junction of an A-form region (first five base pairs near the polymerase active site) and a B-form region (the last nine base pairs toward the RNase H active site). The 41 degrees bend occurs smoothly over the four base pairs between the A-form portion and the B-form portion in the vicinity of helices alpha H and alpha I of the p66 thumb subdomain. The interactions between the dsDNA and protein primarily involve the sugar-phosphate backbone of the nucleic acid and structural elements of the palm, thumb, and RNase H of p66, and are not sequence specific. Amino acid residues from the polymerase active site region, including amino acid residues of the conserved Tyr-Met-Asp-Asp (YMDD) motif and the "primer grip," interact with 3'-terminal nucleotides of the primer strand and are involved in positioning the primer terminal nucleotide and its 3'-OH group at the polymerase active site. Amino acid residues of the "template grip" have close contacts with the template strand and aid in positioning the template strand near the polymerase active site. Helix alpha H of the p66 thumb is partly inserted into the minor groove of the dsDNA and helix alpha I is directly adjacent to the backbone of the template strand. Amino acid residues of beta 1', alpha A', alpha B', and the loop containing His539 of the RNase H domain interact with the primer strand of the dsDNA.

Cross-linking of Engineered Subunit δ to (αβ)3 in Chloroplast F-ATPase

Ser --> Cys mutations were introduced into subunit delta of spinach chloroplast F0F1-ATPase (CF0CF1) by site-directed mutagenesis. The engineered delta subunits were overexpressed in Escherichia coli, purified, and reassembled with spinach chloroplast F1-ATPase (CF1) lacking the delta subunit (CF1(-delta)). By modification with eosin-5-maleimide, it was shown that residues 10, 57, 82, 160, and 166 were solvent-accessible in isolated CF1 and all but residue 166 also in membrane-bound CF0CF1. Modification of the engineered delta subunit with photolabile cross-linkers, binding of delta to CF1(-delta), and photolysis yielded the same SDS gel pattern of cross-link products in the presence or absence of ADP, phosphate, and ATP and both in soluble CF1 and in CF0CF1. By chemical hydrolysis of cross-linked CF1, it was shown that deltaS10C was cross-linked within the N-terminal 62 residues of subunit beta. deltaS57C, deltaS82C, and deltaS166C were cross-linked within the N-terminal 192 residues of subunit alpha. Cross-linking affected neither ATP hydrolysis by soluble CF1 nor its ability to reassemble with CF0 and to structurally reconstitute ATP synthesis. Functional reconstitution, however, seemed to be impaired.

The role of conserved histidine residues in the pyridine nucleotide transhydrogenase of Escherichia coli.

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+, coupled to translocation of protons across the cytoplasmic membrane. The role of histidine residues in catalysis was investigated by chemical modification with diethylpyrocarbonate and by site-directed mutagenesis. Diethylpyrocarbonate inhibited both hydride ion transfer and coupled proton translocation. Histidine residues were modified as shown spectroscopically and by the ability of hydroxylamine to cause reversal of inhibition. Complete inhibition of hydride ion transfer occurred following modification of 10 residues/enzyme molecule. Site-directed mutagenesis of single conserved histidine residues or the presence of substrates did not provide resistance to inhibition by diethylpyrocarbonate. It is concluded that diethylpyrocarbonate inhibition was a consequence of the structural changes brought about by modification of many histidine residues. With the exception of beta-subunit residue His91 (beta His91), in which mutation can result in specific loss of proton translocation activity [Glavas, N. A., Hou, C. & Bragg, P. D. (1995) Biochemistry 34, 7694-7702], site-directed mutation of the remaining conserved residues alpha His450, beta His161, beta His345 and beta His354 did not demonstrate a direct role for these residues in catalysis. Mutation of beta His161 had relatively little effect on the properties of the enzyme. By contrast, mutation of alpha His450, beta His345 and beta His354 caused major loss of enzyme activities which was probably due to alterations in the structure of the enzyme. These alterations were reflected in changes in the K(m) values for transhydrogenation.

Tyrosine phosphorylation of Shc is not required for proliferation or viability signaling by granulocyte-macrophage colony-stimulating factor in hematopoietic cell lines.

The receptor for human granulocyte-macrophage (GM)-CSF (GMR) is a heterodimer, consisting of an alpha-chain (GMR alpha) and a beta-chain (GMR beta). While GMR alpha is capable of binding GM-CSF, GMR beta is necessary for signal transduction. Phosphorylation of one or more tyrosine residues in GMR beta is an early event in signaling. We have recently demonstrated that tyrosine 750 (Y750) in GMR beta is a site of GM-CSF-induced phosphorylation and this site may contribute to the maintenance of cellular viability in response to GM-CSF. To investigate possible contributions made by additional GMR beta cytoplasmic tyrosine residues to receptor function, we mutated other selected tyrosine residues to phenylalanine and tested for any defects in signaling. in the present study, we show that Y577 is required for phosphorylation of Shc and an Shc-associated p140 in response to GM-CSF. Y577 is also required for association of Shc with GRB2. Y577 does not appear to be necessary for GM-CSF-induced proliferation and survival. GMR beta with a mutated Y577 is able to transduce signals leading to the activation of the Raf-1 pathway and the Jak-Stat pathway. Interestingly, mutation of Y750 reduced detectable GM-CSF-induced tyrosine phosphorylation of GMR beta, suggesting that the reduction of Shc phosphorylation associated with that mutant might be actually due to a failure to phosphorylate Y577. These data indicate that the phosphorylation of Shc in response to GM-CSF is not required for proliferation or viability signaling in these cells.

Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (alpha 2 beta beta') and the holoenzyme (core+sigma 70) shows that absence of the sigma 70 subunit is associated with the appearance of several cleavage sites on the subunits beta (within 10 residues of sequence positions 745, 764, 795, and 812) and beta' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near beta residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when sigma 70 binds. No differences are observed for the alpha subunit.

Intersubunit Surfaces in G Protein αβγ Heterotrimers: ANALYSIS BY CROSS-LINKING AND MUTAGENESIS OF βγ

Heterotrimeric guanine nucleotide binding proteins (G proteins) are made up of alpha, beta, and gamma subunits, the last two forming a very tight complex. Stimulation of cell surface receptors promotes dissociation of alpha from the beta gamma dimer, which, in turn, allows both components to interact with intracellular enzymes or ion channels and modulate their activity. At present, little is known about the conformation of the beta gamma dimer or about the areas of beta gamma that interact with alpha. Direct information on the orientation of protein surfaces can be obtained from the analysis of chemically cross-linked products. Previous work in this laboratory showed that 1,6-bismaleimidohexane, which reacts with cysteine residues, specifically cross-links alpha to beta and beta to gamma (Yi, F., Denker, B. M., and Neer, E. J. (1991) J. Biol. Chem. 266, 3900-3906). To identify the residues in beta and gamma involved in cross-linking to each other or to alpha, we have mutated the cysteines in beta 1, gamma 2, and gamma 3 and analyzed the mutated proteins by in vitro translation in a rabbit reticulocyte lysate. All the mutants were able to form beta gamma dimers that could interact with the alpha subunit. We found that 1,6-bismaleimidohexane can cross-link beta 1 to gamma 3 but not to gamma 2. The cross-link goes from Cys25 in beta 1 to Cys30 in gamma 3. This cysteine is absent from any of the other known gamma isoforms and therefore confers a distinctive property to gamma 3. The beta subunit in the beta 1 gamma 2 dimer can be cross-linked to an unidentified protein in the rabbit reticulocyte lysate, generating a product slightly larger than cross-linked beta 1 gamma 3. The beta subunit can also be cross-linked to alpha, giving rise to two products on SDS-polyacrylamide gel electrophoresis, both of which were previously shown to be formed by cross-linking beta to Cys215 in alpha o (Thomas, T. C., Schmidt, C. J., and Neer, E. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10295-10299). Mutation of Cys204 in beta 1 abolished one of these two products, whereas mutation of Cys271 abolished the other. Because both alpha-beta cross-linked products are formed in approximately equal amounts, Cys204 and Cys271 in beta are equally accessible from Cys215 in alpha o. Our findings begin to define intersubunit surfaces, and they pose structural constraints upon any model of the beta gamma dimer.

DNA polymerase betas from liver and testes of cherry salmon, Oncorhynchus masou: purification and characterization of DNA polymerase betas with acidic isoelectric points.

DNA polymerase betas from cherry salmon, Oncorhynchus masou, liver and testes were purified to near homogeneity, and no substantial differences between the enzymes were observed. The molecular weight of both enzymes, determined by SDS-polyacrylamide gel electrophoresis, was 39,000. The amino acid sequences of the N-terminus of the liver and testes enzymes were determined and compared with that of the rat enzyme. Of the N-terminal 30 amino acid residues of salmon liver DNA polymerase beta, 21 (70%) were identical to those of the rat enzyme sequence. However, unlike most eukaryotic DNA polymerase betas, the isoelectric points (pIs) of the DNA polymerase betas from salmon liver and testes were both estimated to be 6.2, which is significantly different from the alkaline isoelectric points (pI = 8.5-9.5) established for other highly purified vertebrate DNA polymerase betas. The cherry salmon DNA polymerase betas were still active at below 10 degrees C, compared with the rat enzyme.

Selective biochemical modification of functional residues in recombinant human macrophage colony-stimulating factor beta (rhM-CSF beta): identification by mass spectrometry.

A rapid method combining classical chemical modification with mass spectrometry was developed to identify amino acids in the recombinant human macrophage colony-stimulating factor (rhM-CSF) protein of potential import to the ligand-receptor interaction. Diethyl pyrocarbonate modification of rhM-CSF beta (under nondenaturing conditions) results in a time- and concentration-dependent loss in receptor binding and biological activity. Peptide mapping of the reaction products by mass spectrometry showed that, with low DEP:M-CSF ratios (< 50:1), there was selective modification of histidine residues, whereas at higher ratios (> 50:1), Tyr and Lys residues were also modified. The loss in rhM-CSF beta activity was directly correlated with the extent of carbethoxylation of His9 and His15, as determined by matrix-assisted laser desorption/ionization mass spectrometric molecular weight determinations (MALDIMS). For these residues mono-modification was observed. By contrast, C-terminal histidine residues His176 and His210 showed bis-modifications, the extent of which had no correlation to losses in biological activity. These data suggest the importance of residues in the A-helix (His9 and His15) to ligand-receptor binding.

Strategy for recombinant multichain protein synthesis: fibrinogen B beta-chain variants as thrombin substrates.

Thrombotic disease has been found in patients with congenital dysfibrinogens that have abnormalities in the amino terminal domain of the fibrinogen B beta-chain. Surprisingly, these fibrinogens are poor substrates for thrombin. In order to examine the molecular basis for this impaired thrombin-fibrinogen interaction, we synthesized three fibrinogens with single amino acid substitutions in this domain: B beta A68T, B beta P70S, and B beta L72S. B beta-chain expression vectors were altered by PCR-directed mutagenesis of the B beta cDNA. The altered vectors were transfected into a Chinese hamster ovary (CHO) cell line that was constructed as a first step in recombinant fibrinogen synthesis, this CHO line synthesizes fibrinogen A alpha- and gamma-chains. More than 86% of the stably selected clones expressed significant levels of fibrinogen, confirming that a two-step strategy permitted efficient synthesis of variant fibrinogens. In large-scale cultures variant fibrinogen accumulation in serum-free medium fluctuated between 1 and 15 micrograms/mL. Normal and variant recombinant fibrinogens were compared to plasma fibrinogens by following the time course of thrombin-catalyzed release of fibrinopeptides. Only the variant B beta A68T, a change identified in a congenital dysfibrinogen, showed significantly impaired kinetics. The rate of fibrinopeptide A release was decreased 27-fold, and the rate of fibrinopeptide B release was decreased 45-fold relative to normal fibrinogen. Fibrinopeptide release was not significantly altered by the substitutions B beta P70S or B beta L72S. These results suggest that B beta residue Ala68 has a novel and critical role in the interaction between thrombin and fibrinogen.

Follitropin conformational stability mediated by loop 2 beta effects follitropin-receptor interaction.

Follicle-stimulating hormone (FSH) is in the family of pituitary/placental glycoprotein hormones which also includes luteinizing hormone (LH), chorionic gonadotropin (hCG), and thyroid-stimulating hormone. These hormones are heterodimers composed of common alpha- and similar but unique beta-subunits. The 21 amino acid loop between Y33 and F53 of the FSH beta-subunit (L2 beta) can be switched into L2 beta of hCG beta without a loss of receptor binding, yet mutation of hFSH beta 37LVY39 to 37AAA39 was antecendent to a 20-fold reduction in receptor binding (based on ID50). A mutation in the LH beta gene, which causes Q54 to be R, causes hypogonadism. This residue is conserved in the glycoprotein hormones and corresponds to Q48 in hFSH beta. Mutation of hFSH beta 48QKTCT52 to 48AAACA52 resulted in a failure of heterodimer formation. In the current study single mutations were made to pinpoint which of the seven hFSH beta residues in the 37LVY39 to 37AAA39 and the 48QKTCT52 to 48AAACA52 mutants were responsible for the observed phenotypes. A single mutation of T52 to alanine was sufficient to cause a reduction in expression of heterodimeric hormone. Single mutants Q48A, T50A, V38A, Y39A, and, to a lesser extent, T52A formed heterodimer. However, these hFSH mutants were markedly unstable at pH 2.0. Thus, acid dissociation can be used to reveal metastable forms of this protein. Mutant hFSH beta Q48A was also 8-fold less active than wild-type hFSH when assayed for binding to hFSH receptors. hFSH beta V38A and Y39A mutants affected receptor binding; however, neither mutation alone caused greater than a 2-fold decrease in receptor binding activity. In summary, these results identify single important residues in the long loop (between Y33 and F53) of the hFSH beta-subunit which are required for proper subunit interactions that provide conformational stability which in turn is necessary for FSH-receptor interaction.

Purification and partial amino acid sequence of plantaricin S, a bacteriocin produced by Lactobacillus plantarum LPCO10, the activity of which depends on the complementary action of two peptides.

Plantaricin S, one of the two bacteriocins produced by Lactobacillus plantarum LPCO10, which was isolated from a green-olive fermentation (R. Jiménez-Díaz, R.M. Ríos-Sánchez, M. Desmazeaud, J.L.Ruiz-Barba, and J.-C. Piard, Appl. Environ. Microbiol. 59:1416-1424, 1993), has been purified to homogeneity by ammonium sulfate precipitation, by binding to SP-Sepharose fast-flow, phenyl-Sepharose CL-4B, and C2/C18 reverse-phase chromatographies. The purification resulted in a final yield of 91.6% and a 352,617-fold increase in the specific activity. The bacteriocin activity was associated with two distinct peptides, termed alpha and beta, which were separated by C2/C18 reverse-phase chromatography. Although beta alone appeared to retain a trace of inhibitory activity, the complementary action of both the alpha and beta peptides was required for full bacteriocin activity, as judged by both the agar well diffusion and the microtiter plate assays. From the N-terminal end, 26 and 24 amino acids residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acids residues, suggesting that either modifications in the next amino acid residue blocked the sequencing region or that the C-terminal end had been reached. The amino acid sequences of alpha and beta show no apparent homology to each or to other bacteriocins purified from lactic acid bacteria.